This invention is directed to CDNA sequences, cell lines, and polypeptides containing epitope(s) for high-affinity antibodies within the A-beta fragment of the beta-amyloid precursor protein (beta-APP). A preferred embodiment of the invention relates to assays useful for identifying and characterizing beta amyloid precursor processing inhibitors, and for purifying APP processing enzymes. Such assays are useful in developing beta-APP processing modulators for the treatment of diseases associated with altered metabolism of beta-APP and an accumulation of amyloid A-beta.
The present invention is directed generally to methods and compositions for monitoring the processing of epitope-tagged beta-APP. More specifically, the present invention relates to the use of such methods and compositions for monitoring responses of cells expressing such epitope-tagged beta-APP, or fragments thereof. The present invention also relates to cell free systems containing the epitope-tagged polypeptides, therapy of diseases associated with an altered metabolism of the beta-amyloid precursor protein (beta-APP), and for screening and evaluation of potential drugs for the treatment of these disorders, including Alzheimer""s disease.
Alzheimer""s disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, temporal and local orientation, cognition, reasoning, judgment and emotional stability. AD is a common cause of progressive dementia in humans and is believed to be one of the major causes of death in the United States. AD has been observed in all races and ethnic groups worldwide and presents a major present and future health problem. No treatment that effectively prevents AD, or reverses the clinical symptoms and underlying pathophysiology is currently known.
Histopathological examination of brain tissue obtained upon autopsy, or from neurosurgical specimens in affected individuals, revealed the occurrence of amyloid plaques and neurofibrillar tangles in the cerebral cortex of such patients. Similar alterations were observed in patients with Trisomy 21 (Down""s syndrome), and hereditary cerebral hemorrhage with amyloidosis of the Dutch-type. Neurofibrillar tangles are nonmembrane-bound bundles of abnormal proteinaceous filaments. Biochemical and immunochemical studies led to the conclusion that their principle protein subunit is an altered phosphorylated from of the tau protein (reviewed in Selkoe, D. J. Annu Rev Cell Biol 1994 10: 373-403).
Biochemical and immunological studies show that the dominant proteinaceous component of the amyloid plaque is a 4.2 kilodalton (kD) protein of from about 39 to about 43 amino acids in length. This protein was designated A-beta, beta-amyloid peptide, or beta/A4. In addition to its deposition in amyloid plaques, A-beta is also found in the walls of meningeal and parenchymal arterioles, small arteries, capillaries, and occasionally venules. A-beta was first purified and a partial amino acid sequence reported in 1984 (Glenner and Wong, Biochem. Biophys. Res. Commun. 120: 885-890). The isolation and sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829, the contents of which are herein incorporated by reference in their entirety.
Compelling evidence accumulated during the last decade revealed that A-beta is an internal polypeptide derived from a type 1 integral membrane protein, termed beta-APP. Beta-APP is normally produced by many cells both in vivo and in cultured cells derived from various animals and humans. A-beta is derived from cleavage of beta-APP by an as yet unknown enzyme (protease) system(s), collectively termed secretases. The existence of at least three proteolytic activities has been postulated. They include (a) beta secretase(s), generating the N-terminus of A-beta, (b) alpha secretase(s) cleaving in the region of the 16/17 peptide bond in A-beta, and (c) gamma secretases, generating C-terminal A-beta fragments ending at position numbers 38, 39, 40, 42, and 43. The precise biochemical mechanism by which A-beta is derived from beta-APP, and how it subsequently accumulates in cerebral tissue and blood vessels, is currently unknown.
Several lines of evidence suggest that the abnormal accumulation of A-beta plays a key role in the pathogenesis of AD. First, A-beta is the major protein found in amyloid plaques (Glenner G G, Wong, C W 1984 Biochem Biophys Res Commun 120: 885-90). Second, A-beta is neurotoxic and may be causally related to neuronal death observed in AD patients (Pike C J, Burdick, D, Walencewicz A J, Glabe C G, Cotman C W 1993 J Neurosci 13: 1676-87). Third, missense DNA mutations at position 717 in the 770 isoform of beta-APP can be found in affected members but not in unaffected members of several families with a genetically determined (familial) form of AD (Goate A, Chartier-Harlin M-C, Mullan M, Brown J, Crawford F et al 1991 Nature 349: 704-6). In addition, several other beta-APP mutations have been described in familial forms of AD (reviewed in Selkoe D J 1994 Annu Rev Cell Biol 1994 10: 373-403). Fourth, similar neuropathological changes have been observed in transgenic animals overexpressing mutant forms of human beta-APP (Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G 1996 Nature 274: 99-102). Finally, individuals with Down""s syndrome have an increased gene dosage of beta-APP and develop early-onset AD. Taken together, these observations strongly suggest that A-beta depositions may be causally related to Alzheimer""s disease.
While large progress has been made in understanding the underlying cause(s) of AD and other A-beta related diseases, there remains a need to develop methods and compositions for the treatment of the disease(s). Treatment methods could be based on compounds that inhibit the formation of A-beta in vivo. To identify and characterize such compounds, high-throughput screening methods are required to identify compounds that affect beta-APP processing. Specific assays for A-beta detection should be able to detect A-beta in biological samples at very low concentrations, as well as distinguishing between A-beta and other fragments of beta-APP that may be present in the sample.
U.S. Pat. No. 4,666,829, suggests the use of an antibody to the 28-amino acid fragment of A-beta to detect xe2x80x9cAlzheimer""s Amyloid Polypeptidexe2x80x9d in biological samples. This suggestion was not used in the present invention.
Several attempts to measure A-beta in biological samples by immunological methods have been reported. While these studies detect very low levels of A-beta peptides, no attempts to purify and characterize this immunoreactivity further and to determine whether it indeed represents A-beta have been reported.
U.S. Pat. No. 5,593,846 describes assays aimed at determining A-beta levels in biological samples using antibodies based on the native A-beta sequence. No attempt was made to differentiate between A-beta peptides with heterogenous C-termini by either enzyme-linked immunosorbant assay (ELISA), or radioimmunoassay (RIA). More specifically, the assay system described therein is based on antibodies that recognize epitopes in the A-beta polypeptide between amino acids 1 to 28 of A-beta, and does not differentiate between A-beta (1-40) and A-beta (1-42). In addition, the assay is not anticipated to be sufficiently sensitive to detect the levels of A-beta (1-42) that are expected to accumulate in beta-APP transfected mammalian cells in the 96-well microtiter plate format required for high-throughput drug screening.
While mutations have been introduced within the A-beta sequence of beta-APP (for example Citron M, Teplow D B, Selkoe D J 1995 Neuron 14:661-670), the use of beta-APP molecules epitope-tagged within the A-beta sequence has not been explored.
The present invention provides methods that enable one skilled in the art to identify and characterize beta amyloid production inhibitors. Recombinant expression constructs were prepared containing an epitope for a high-affinity antibody centered about the alpha secretase cleavage site of the A-beta fragment of the beta-APP. Cell lines were established that express these mutant forms of beta-APP and secrete fragments of epitope-tagged beta-APP. In addition, epitope-tagged beta amyloid fragments were also prepared by chemical synthesis.
An object of the invention provides assays and methods for the detection and characterization of epitope-tagged beta-APP and fragments thereof. The invention provides enzyme-linked immunosorbent assays (ELISA) and other immunological methods suitable for the detection of alpha, beta, and gamma secretase modulators in cell-based and cell-free assay systems. An accumulation of epitope-tagged A-beta was observed in cells transfected with the expression cassette. The cultured cells were exposed to test compounds that cause a change in the secreted amount of soluble epitope tagged fragments of beta-APP. The present assays and methods may be used to identify beta amyloid precursor processing modulators and to develop therapeutic modalities based on modification of beta amyloid precursor processing.